Conditioned surfaces for in situ molecular array synthesis

ABSTRACT

Described herein are in situ synthesized arrays and methods of making the them, wherein array signal sensitivity and robustness is enhanced by carrying out conditioning steps and/or generating linkers during synthesis. An array comprises a surface with a collection of features, wherein the features comprise molecules or polymers attached to the surface. In certain embodiments of the invention, carrying out conditioning steps during array synthesis can yield arrays with improved signal. In other embodiments, linkers are synthesized on the array surface prior to synthesis of functional molecules, wherein increasing linker length can correspond to an improvement in the signal generated by the array.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/264,426, filed Sep. 13, 2016 with claims the benefit of U.S.Provisional Application No. 62/218,418, filed Sep. 14, 2015, each ofwhich is incorporated herein by reference in its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under MCB-1243082awarded by the National Science Foundation. The government has certainrights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 28, 2016, isnamed 42206-708_201_SL.txt and is 3,456 bytes in size.

BACKGROUND

Array technologies allow for large-scale, quantitative analyses ofbiological samples. However, the density and robustness of suchtechnologies should be improved to allow increased sensitivity,reproducibility and accuracy of biological assays using such arrays.

SUMMARY OF THE INVENTION

Disclosed herein are methods and devices for making an array, the methodcomprising (a) performing a conditioning step on a surface of the arrayin the absence of monomer; (b) repeating step (a) at least once; (c)performing a synthesis step upon the surface to add at least onemonomer; and (d) repeating step (c) at least once to form a sequence,wherein the conditioning step performed prior to synthesizing thesequence enhances attachment of the sequence to the surface of thearray.

In some embodiments, the method comprises the conditioning step to beperformed at least 5 times, at least 10 times or at least 15 times. Inother embodiments, the binding of a target to a sequence is enhanced bythe methods and devices disclosed herein by least 10%, at least 20%, atleast 30%, at least 40% or at least 50%. In yet other embodiments, thebinding of a target to a sequence is enhanced by the methods and devicesdisclosed herein by least 1-fold, at least 2-fold, at least 3-fold, atleast 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold. Instill other embodiments, the sensitivity of the array is enhanced by themethods and devices disclosed herein by at least 10%, at least 20%, atleast 30%, at least 40% or at least 50%. In still other embodiments, thesensitivity of the array is enhanced by the methods and devicesdisclosed herein by at least 1-fold, at least 2-fold, at least 3-fold,at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, atleast 8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.

Also disclosed herein are methods and devices for making an array,comprising (a) performing a chemical reaction upon the array that iscapable of attaching a monomer to a coupling site, wherein the monomerforms part of a linker sequence; (b) performing an additional chemicalreaction upon the array that is capable of adding a monomer to theattached monomer of step (a); (c) repeating step (b) at least once untilthe monomers that comprise the desired linker sequence have been added;and d. synthesizing in situ a functional sequence onto the free end ofthe linker sequence, wherein the linker sequence enhances bindingbetween the functional sequence and a substrate.

In some embodiments, the methods and devices disclosed herein to make anarray include a linker sequence between 2 and 5 monomers, between 6 and10 monomers, between 11 and 15 monomers, between 16 and 20 monomers,between 21 and 25 monomers, between 26 and 30 monomers, or at least 30monomers. In yet other embodiments, the linker sequences is at least 10%homogeneous, at least 20% homogeneous, at least 30% homogeneous, atleast 40% homogeneous, at least 50% homogeneous, at least 60%homogeneous, at least 70% homogeneous, at least 80% homogeneous, atleast 90% homogeneous, or at least 100% homogeneous.

In still other embodiments, the linker sequence has a net negativecharge at a pH of 7. In other embodiments, the linker sequence has a netpositive charge at a pH of 7. In yet other embodiments, the linkersequence has a neutral charge at a pH of 7.

In other embodiments, the linker sequence is composed of moleculesselected from the group consisting of nucleotides, oligonucleotides,polynucleotides, ribonucleotides, oligoribonucleotides,polyribonucleotides, monosaccharides, oligosaccharides, polysaccharides,amino acids, peptides, polypeptides, proteins, lipids, synthetic ornon-natural compounds, and peptide nucleic acids (PNAs). In additionalembodiments, the linker sequence is composed of amino acids selectedfrom the group consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, selenocysteine, pyrrolysine,N-formylmethionine, L-theanine, gamma amino butyric acid (GABA),ornithine, citrulline, α-methylnorvaline, β-alanine, δ-aminolevulinicacid, and 4-aminobenzoic acid (PABA). In other embodiments, the linkersequence is homogeneously composed of glycine. In yet other embodiments,the linker sequence comprises at least one monomer unit of polyethyleneglycol (PEG), diamines, diacids, amino acids, alcohols, thiols, orcombinations thereof.

Also disclosed herein are arrays comprising: a. a surface, and b. aplurality of molecules, wherein the molecules comprise a linker sequencecomprised of monomers and having two ends, wherein one end is coupled tothe surface and the other end is coupled to a functional sequence whichbinds a target molecule. Also disclosed herein are arrays comprising:(a) a surface, and (b) a plurality of in situ synthesized moleculesimmobilized to the surface, wherein each molecule comprises a linkerattached to a functional sequence, the linker being at least 1 monomerin length and interposed between the surface of the array and thefunctional sequence, and the functional sequence being at least 2monomers in length.

Disclosed herein are peptide arrays comprising: (a) a surface, and (b) aplurality of peptides immobilize to the surface, wherein each peptidecomprises a linker sequence attached to a functional sequence, thelinker sequence being interposed between the surface of the array andthe functional sequence. Also disclosed herein are methods of making anarray of molecules, comprising: a. performing a first chemical reactionon the array, wherein a pre-synthesized linker is attached to a surfaceof the array; and b. performing a second chemical reaction upon thearray, wherein a pre-synthesized functional sequence is attached to thelinker sequence, wherein the linker sequence enhances binding betweenthe functional sequence and a target.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 illustrates the results of the surface stability test performedupon peptides synthesized successively. The y-axis is binding strength.Along the x-axis are the sequences of peptides synthesized. The lowercase letters (a,b,c,d) represent the order in which the monoclonalantibody epitope RHSVV sequences (SEQ ID NO: 1) were synthesized. Thus,the sequence RHSVVa (SEQ ID NO: 1) was synthesized first and wassubjected to all of the conditions associated with synthesizing RHSVVb(SEQ ID NO: 1), RHSVVc (SEQ ID NO: 1) and RHSVVd (SEQ ID NO: 1). As canbe seen, sequences made later in the synthesis resulted in greaterbinding, presumably because there was less opportunity of removal ormodification. The Ab1 epitopes were on aminosilane surface withoutstabilization cycles.

FIG. 2 is similar to FIG. 1 in terms of axis definitions. It illustratesthe results of the surface stability test performed upon peptidessynthesized successively on a seven amino acid linker (SEQ ID NO: 5).The Ab1 epitopes (all disclosed as SEQ ID NO: 8) were synthesized 4times, labeled with a, b, c, d, on aminosilane surface after 20stabilization cycles; the linkers are synthesized during thestabilization cycles.

FIG. 3 is similar to FIG. 1 in terms of axis definitions. It illustratesthe results of the surface stability test performed upon peptidessynthesized successively on a three amino acid linker. The Ab1 epitopes(all disclosed as SEQ ID NO: 7) were synthesized 4 times, labeled witha, b, c, d, on aminosilane surface after 20 stabilization cycles; thelinkers are synthesized during the stabilization cycles.

FIG. 4 illustrates the results of the surface stability test as afunction of the length and composition of various linkers. The Ab1epitopes were synthesized 4 times, labeled with a, b, c, d, on anaminosilane surface after 20 stabilization cycles; the linkers aresynthesized during the stabilization cycles. Figure discloses SEQ ID NOS7, 9-11, 8, 12, 7, 9-11, 8, 12, 7, 9-11, 8, 7, 9-11, 8, 12, 13, 2-6 and14, respectively, in order of appearance.

FIG. 5 is a visual representation of an immunosignature as a heat map.The x axis represents specific peptide features on the array used. The yaxis represents specific samples applied to the array. The samples weretaken from individuals that had Dengue fever (Dengue), Malaria, WestNile Virus (WNV) or had no disease (ND). The top sample was a control inwhich the fluorescently labeled secondary antibody used to detect IgGbinding in the other samples is used in the absence of a blood sample.

DETAILED DESCRIPTION OF THE INVENTION

Arrays can be generated using methods that include immobilizing purifiedmolecules or monomers onto suitable surfaces or synthesizing them insitu directly on the surface. In situ synthesis offers significantadvantages over other methods such as a greater number and density offeatures, and enhanced sensitivity, but the quality and reliability ofin situ arrays can be contingent on the quality and consistency of thesynthesized molecules. One important aspect of an in situ syntheticprocess is the preparation of the surface upon which in situ synthesisis performed. Factors that are important to in situ array synthesis caninclude the proper preparation of the surface, the availability ofattachment groups that can be used as a base for attachment, the densityof those groups, the tendency of the surface or matrix surrounding theattachment groups to interfere with the function of the finalsynthesized molecule, for example through nonspecific binding or bymodifying the synthesized molecule to interact with a molecular partnerof choice, and the evenness of the distribution of attachment groups ofmultiple length.

Different methods exist for placing attachment groups on surfaces. Oneof the most common surface modifications, particularly for surfaces thathave a large fraction of silicon oxide, is to use a functionalizedsilane, for example an amino silane. Depending on the chemistry ofattachment, such molecules can add to the surface in a well-definedlayer or as a more complex cross-linked structure. In situ synthesissteps can then be performed upon the attachment groups on the surface byexposing them to monomers in reactive coupling steps that link themtogether.

The present disclosure recognizes the advantages of preparingconditioned surfaces for in situ molecular array synthesis. Some arrays,such as the arrays described in PCT/US2013/065541 and U.S. Pat. No.5,424,186, hereinafter incorporated by reference in their entireties,provide a method of analyzing various materials including biologicalsamples. However, the sensitivity and consistency of array-baseddetection can depend upon the properties of the attachment groups andthe surface on which they reside.

Recognized herein is that molecules of previously described arrays arenot all equally available for binding on the array. Such variability canlead to differences in sensitivity between features on or betweenarrays, and thus reproducibility of biological assay results on thesearrays. The present disclosure recognizes that compensation can be madefor this variability by increasing or growing a linker molecule ormolecules on the attachment sites. Accordingly, included herein aremethods to modify or increase the length of linkers on attachment sitesof the arrays in order to optimize binding of targets in the biologicalsamples to molecules attached to the array.

Also recognized herein is that the attachment groups of previouslydescribed arrays are not all equally available for reactive coupling.Moreover, the present disclosure recognizes that the surface material ofan array is not entirely functionally inert (both in terms of thesynthesis and the final function of the molecule) at locations otherthan the chemical attachment site. The present disclosure alsorecognizes that the structure of the silane or other attachment matricescan be affected by the synthetic process. As a consequence, some in situsynthesis methods may lead to the generation of surfaces with differentproperties in earlier synthetic steps versus later synthetic steps.

Described herein are methods for in situ synthesis of arrays thatsignificantly enhances attachment of a sequence to the surface of thearray. These methods allow for the production of arrays with enhancedsensitivity that are applicable to a wide variety of applications,including but not limited to: diagnostic arrays, arrays for selection ofspecific ligands or targets, arrays for potential drugs, and arrays ofsensor molecules. In some cases, the approach can comprise performingmultiple cycles (or partial cycles) of surface conditioning prior to themain synthesis, thereby allowing a surface to become stabilized beforethe molecules or sequences are actually in situ synthesized, orotherwise incorporated, into the array. The result can be an arraywherein the molecules or sequences bind to their ligands or targets withgreater sensitivity and reduced background noise.

In some cases, the cycles are run in such a way that no coupling of anew molecular component to the system can occur prior to the mainsynthesis. In other cases, the cycles are run in such a way thatcoupling of a new molecular component to the system can occur prior tothe main synthesis. In yet other cases, the process involves acombination of cycles that couple new molecular components to the systemand cycles that do not couple new molecular components to the systemprior to the main synthesis. In some instances, the process involvesgrowing a “linker” on the attachment sites that is attached to amolecule or sequence prior to the main synthesis.

Also provided herein is method of providing a conditioned surface to anarray. In some cases, the surface comprises a number of in situsynthesized molecules attached to a conditioned surface. The surface cancomprise a number of molecules that have been synthesized in situ orotherwise added to the array.

Definitions

Terms that are not defined in the present application or anyincorporated references will be given their plain and ordinary meaningin the field as understood by one of ordinary skill in the art.

As used in the present specification, the following words, phrases andsymbols are generally intended to have the meanings as set forth below,except to the extent that the context in which they are used indicatesotherwise. The following abbreviations and terms have the indicatedmeanings throughout:

As used in the specification and claims, the singular forms “a”, “an”and “the” include plural references unless the context clearly dictatesotherwise. For example, the term “an array” may include a plurality ofarrays unless the context clearly dictates otherwise.

An “array” refers to an intentionally created collection of molecules orsequences attached to or fabricated on a substrate or surface in whichthe identity or source of a group of molecules is known based on itslocation on the array. The molecules or sequences housed on the arrayand within a feature of an array can be identical to or different fromeach other.

Molecules can include nucleotides, oligonucleotides, polynucleotides,ribonucleotides, oligoribonucleotides, polyribonucleotides,monosaccharides, oligosaccharides, polysaccharides, amino acids,peptides, polypeptides, proteins, lipids, synthetic or non-naturalcompounds, peptide nucleic acids (PNAs) and other materials known in theart. In some embodiments, the molecules are a plurality of monomers thatform a polymeric sequence. In other embodiments, the polymeric sequencesbind to or recognize a target, e.g., ligand or binding partner, in asample or specimen. In yet other embodiments, the molecules or polymericsequences may include a linker as described herein.

Monomers can include nucleotides, monosaccharides, amino acids, andother subunit components. Monomer molecules can include natural aminoacids, non-natural amino acids, nucleic acids, and analogues thereof.

A polymer can comprise two or more linked monomers. The monomers in aparticular polymeric linker are not necessarily identical and can be ofheterogeneous composition, comprising different monomers of the sameclass of molecules.

A class of molecules can be each of the following: nucleotides,oligonucleotides, polynucleotides, ribonucleotides,oligoribonucleotides, polyribonucleotides, monosaccharides,oligosaccharides, polysaccharides, amino acids, peptides, polypeptides,proteins, lipids, synthetic or non-natural compounds, or peptide nucleicacids (PNAs).

Polymers can be copolymers such as alternating copolymers, periodiccopolymers, statistical copolymers, or block copolymers. Polymers cancomprise linear or branched structures. Branched structures can resultwhen one or more substituents or side groups on a parent polymeric chainare replaced by another chain of a polymer. The substituted polymericchain or branch or side chain can be comprised of the same class orclasses of monomers, or of different class or classes of monomers fromthe parent polymeric chain. In some branched structures, the side chainscan grow off the parent chain at regular intervals such as every 10monomeric subunits. In others, the side chains can grow off the parentchain at irregular intervals. A branched polymer can be a graft polymer,a star-shaped polymer, a comb polymer, or a dendrimer. Polymers can alsoform a polymer network in which all polymer chains are interconnectedthrough branching and/or crosslinking to form a macroscopic entity.Crosslinking can occur between branches on the same polymer or betweenbranches of distinct polymers. Where linear and branched polymers are inproximity, crosslinking can also occur between those linear and branchedpolymers. The three-dimensional structure of a polymer can be lacking adefined structure. For example, the polymer may be a flexible loop thatlacks active sites capable of bonding or interacting with other sites onthat same loop or with other molecules, which can cause its orientationand shape to continually change. Conversely, a polymer can form a moredefined structure. For example, a peptide polymer may interact withitself or with neighboring polymers to form secondary, tertiary, and/orquaternary structures.

Main synthesis can comprise the synthesis steps that couples monomers toan attachment group on the surface or to other molecules already coupledto the attachment group. The added monomer can comprise a functionalsequence or an inert sequence.

A “linker” can be either sequentially or non-sequentially incorporatedinto an array. A linker can be added to a growing polymer, or viceversa. A linker can be a component that elongates the distance betweenthe substrate surface of an array and a molecule, such as a polypeptideor polynucleotide. A “linker sequence” can be a polymer of linkercomponents. A “linker” can mean a linker component (including but notlimited to a nucleotide, an amino acid or a chemical constituent), alinker sequence, and/or a linker polymer.

A “functional sequence” or “functional molecule” can recognize or bindto a target, e.g, ligand or binding partner to the functional sequenceor functional molecule.

A “target” is a ligand or binding partner which can bind or interactwith one or more functional molecules or sequences on the surface of anin situ synthesized array.

Couple or coupling comprises forming a chemical bond between twocomponents, including but not limited to two molecules or sequences, orto a molecule or sequence and a linker, or to a molecule and anattachment site. The coupling moieties includes but is not limited tophosphodiester or amide bonds, ester bonds, thioester bonds, etherbonds, and carbon-carbon bonds.

Attach or attachment is equivalent to couple or coupling. An attachmentsite is a chemical or component that is capable of forming a chemicalbond with another molecule. This includes but is not limited tophosphodiester or amide bonds, ester bonds, thioester bonds, etherbonds, and carbon-carbon bonds.

A surface can be a “solid support,” “support,” and “substrate,” thatserves as a physical support for a polymer or a group of polymers.

Linkers

A molecule can be attached to a surface directly or via a linker asdisclosed herein. Direct attachment is possible by covalent attachmentof a molecule, such as an amino acid to a region or feature of thesurface. For example, the linker(s) as disclosed herein could beattached to a chemically reactive or chemically inert region of thesurface. Optionally or additionally, the linkers may be linear orbranched, having at least one or a multiplicity of reactive sites forattachment of a molecule as described herein. A linker can be cleavable,non-cleavable, self-immolating, hydrophilic, or hydrophobic. A linker asdisclosed herein can also be flexible or rigid, or a combinationthereof, which may be dependent upon the specific linker sequenceemployed. For example, glycine-rich linkers tend to be flexible, whereasproline-rich linkers tend to form relatively rigid extended structures.

A linker as disclosed herein can be a component that is sequentially ornon-sequentially incorporated into an array. For example, a linker asdisclosed herein may be formed on the surface of an array bysequentially attaching a linker component to an attachment site (e.g.,aminosilane) followed by additional linker components that are added tothe growing polymer. Alternatively, a linker polymer as disclosed hereinmay be prefabricated or pre-synthesized and then attached to the arraysurface without in situ sequential addition of linker molecules.Likewise, as disclosed herein a pre-synthesized molecule or sequence maybe attached to an in situ synthesized linker or a pre-synthesizedlinker. In addition, as disclosed herein a combination of prefabricatedlinker polymers and in situ synthesis of additional linkers may beutilized to generate the finished linker polymer on the array surface.

In some embodiments, a linker as disclosed herein can be divided intoseparate linkers or sub-linkers by an intervening functional molecule orfunctional sequence. For example, a first linker or sub-linker may beattached to the attachment group on the array surface on one end,wherein an intervening functional sequence is joined or attached to thefree end of the first linker, and a second linker may be then joined orattached to the free end of the intervening functional sequence. Asecond intervening functional sequence may then be joined to the freeend of the second linker, and so on and so forth, wherein a polymer isgenerated that comprises a chain of alternating linker sequences andfunctional sequences.

A linker as disclosed herein can comprise a single monomer linkedtogether, or it can comprise two or more linked monomers. Thus, themonomers in a particular polymeric linker are not necessarily identicaland can be of heterogeneous composition, comprising different monomersof the same class. For example, valine and glycine are differentmonomers within the same class of amino acids. Alternatively, linkersmay comprise monomers that belong to different classes. For example, alinker as disclosed herein may comprise amino acids from differentclasses (e.g., acidic and basic amino acids) or amino acids andnucleotides. Additionally, a linker as disclosed herein may alsocomprise modified amino acids, including but not limited to amino acidsmodified with polyethylene glycol, long-chain amine, long-chain alcoholor polyene derivative.

Linkers as disclosed herein can be copolymers such as alternatingcopolymers, periodic copolymers, statistical copolymers, or blockcopolymers. Linkers as disclosed herein can comprise linear or branchedstructures. Branched structures can result when one or more substituentsor side groups on a parent polymeric chain are replaced by another chainof a polymer. The substituted polymeric chain or branch or side chaincan be comprised of the same class or classes of monomers, or ofdifferent class or classes of monomers from the parent polymeric chainthat forms part of the linker. In some branched structures, the sidechains can grow off the parent chain at regular intervals such as every10 monomeric subunits. In others, the side chains can grow off theparent chain at irregular intervals. A branched polymer linker asdisclosed herein can be a graft polymer, a star-shaped polymer, a combpolymer, or a dendrimer. Linkers as disclosed herein can also form apolymer network in which all polymer chains are interconnected throughbranching and/or crosslinking to form a macroscopic entity. Crosslinkingcan occur between branches on the same linker or between branches ofdistinct linkers. Where linear and branched linkers are in proximity,crosslinking can also occur between those linear and branched linkers.The three-dimensional structure of a linker as disclosed herein can belacking a defined structure. For example, the linker as disclosed hereinmay be a flexible loop that lacks active sites capable of bonding orinteracting with other sites on that same loop or with other linkercomponents, which can cause its orientation and shape to continuallychange. Conversely, a linker as disclosed herein can form a more definedstructure. For example, a peptide linker as disclosed herein mayinteract with itself or with neighboring components to form secondary,tertiary, and/or quaternary structures.

The linkers as disclosed herein may be, for example, aryl acetylene,ethylene glycol oligomers containing 2-20 monomer units, 2-10 monomerunits or 1-5 monomer units including but not limited to polyethyleneglycol (PEG), diamines, diacids, amino acids, alcohols, thiols, andcombinations thereof. Examples of diamines include ethylene diamine anddiamino propane. Alternatively, the linkers as disclosed herein may bethe same type as that being synthesized (i.e., nascent polymers), suchas polypeptides and polymers of amino acid derivatives such as forexample, amino hexanoic acids. While most natural amino acids are alphaamino acids, linkers can comprise monomeric subunits selected fromalpha, beta, gamma, delta, and/or epsilon amino acids. Linkers asdisclosed herein may comprise amino acid monomers in either the L or Disomeric forms. Linkers as disclosed herein can include monomericsubunits selected from the 20 standard proteinogenic amino acids:alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine. In some embodiments, the amino acids C, I, T andM, and optionally Q and E, are not included as subunits in the linker.Linkers as disclosed herein may also have monomeric subunits selectedfrom non-standard proteinogenic amino acids such as selenocysteine,pyrrolysine, and N-formylmethionine. Linkers may also have monomericsubunits selected from non-proteinogenic amino acids, which can includeL-theanine, gamma amino butyric acid (GABA), ornithine, citrulline,α-methylnorvaline, β-alanine, S-Aminolevulinic acid, and/or4-Aminobenzoic acid (PABA). Similarly, the linkers may comprise polymersas defined in this specification. For example, linkers as disclosedherein can comprise nucleic acids (DNA or RNA), oligosaccharides orpolysaccharides, lipids, fatty acids, peptide nucleic acids, syntheticcompounds, or analogues thereof.

Linkers as disclosed herein may be of homogeneous or heterogeneouscomposition. For example, in some embodiments a linker as disclosedherein may be a peptide linker homogeneously composed of only alanine,arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid,glycine, histidine, isoleucine, leucine, lysine, methionine,phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine,selenocysteine, pyrrolysine, N-formylmethionine, L-theanine, gamma aminobutyric acid (GABA), ornithine, citrulline, α-methylnorvaline,β-alanine, δ-Aminolevulinic acid, or 4-Aminobenzoic acid (PABA)monomers. In other embodiments, a peptide linker as disclosed herein maybe heterogeneously composed of more than one type of monomer selectedfrom the group consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, valine, selenocysteine, pyrrolysine,N-formylmethionine, L-theanine, gamma amino butyric acid (GABA),ornithine, citrulline, α-methylnorvaline, 3-alanine, δ-Aminolevulinicacid, and 4-Aminobenzoic acid (PABA) monomers. A heterogeneous linker asdisclosed herein composition may be at least of a certain homogeneouspercentage. For example, a linker composed of 50% Alanine, 10% Glycine,10% Isoleucine, 10% Tryptophan, 10% Serine, and 10% Glutamine is 50%homogeneous. The highest percentage component is the homogeneouspercentage.

In some embodiments, a linker as disclosed herein comprises acomposition that is at least 10% homogeneous, 20% homogeneous, 30%homogeneous, 40% homogeneous, 50% homogeneous, 60% homogeneous, 70%homogeneous, 80% homogeneous, 90% homogeneous, or 100% homogeneous.

In some embodiments, a linker as disclosed herein comprises acomposition that is no more than 10% homogeneous, 20% homogeneous, 30%homogeneous, 40% homogeneous, 50% homogeneous, 60% homogeneous, 70%homogeneous, 80% homogeneous, 90% homogeneous, or 100% homogeneous.

In some embodiments, a linker as disclosed herein comprises acomposition that is 10% homogeneous, 20% homogeneous, 30% homogeneous,40% homogeneous, 50% homogeneous, 60% homogeneous, 70% homogeneous, 80%homogeneous, 90% homogeneous, or 100% homogeneous.

The attachment(s) as disclosed herein between linkers or between alinker and an attachment site on an array surface can be via an amidebond, an ester bond, an ether bond, a carbamate bond, a carbon-nitrogenbond, a triazole, a macrocycle, an oxime bond, a hydrazone bond, imminebonds, a carbon-carbon single double or triple bond, a disulfide bond,thioester bond, or a thioether bond. Non-limiting examples of thefunctional groups for attachment include functional groups capable offorming, for example, an amide bond, an ester bond, an ether bond, acarbonate bond, a carbamate bond, or a thioether bond.

Non-limiting examples of functional groups capable of forming such bondsinclude amino groups; carboxylic acid groups, hydroxyl groups, carboxylgroups; aldehyde groups; azide groups; aniline groups, pyrrole groups,nitrile groups, isonitrile groups, aziridine groups, alkyne and alkenegroups; ketones; hydrazides; acid halides such as acid fluorides,chlorides, bromides, and iodides; acid anhydrides, includingsymmetrical, mixed, and cyclic anhydrides; carbonates; carbonylfunctionalities bonded to leaving groups such as cyano, succinimidyl,and N-hydroxysuccinimidyl; hydroxyl groups; sulfhydryl groups; andcompounds and components possessing, for example, alkyl, alkenyl,alkynyl, allylic, or benzylic leaving groups, such as halides,mesylates, tosylates, triflates, epoxides, phosphate esters, sulfateesters, and besylates.

A linker as disclosed herein may have one or more functional groups, forexample, one functional group that is bonded to the surface, and onefunctional group that is bonded to the growing chain (e.g.: peptidechain), and a linking portion between the two functional groups.Non-limiting examples of the linking portion include alkylene,alkenylene, alkynylene, polyenes, sugars, sugar polymers, nucleic acids,nucleic acid polymers, fatty acids, lipids, polyether, such aspolyethylene glycol (PEG), polyester, polyamide, polyamino acids,polypeptides, cleavable peptides, valine-citrulline, substituted phenylderivatives (for example, hydroxy benzoic acid), aminobenzylcarbamates,D-amino acids, and polyamine, any of which being unsubstituted orsubstituted with any number of substituents, such as halogens, hydroxylgroups, sulfhydryl groups, amino groups, nitro groups, nitroso groups,cyano groups, azido groups, sulfoxide groups, sulfone groups,sulfonamide groups, carboxyl groups, carboxaldehyde groups, iminegroups, alkyl groups, halo-alkyl groups, alkenyl groups, halo-alkenylgroups, alkynyl groups, halo-alkynyl groups, alkoxy groups, aryl groups,aryloxy groups, aralkyl groups, arylalkoxy groups, heterocyclyl groups,acyl groups, acyloxy groups, carbamate groups, amide groups, urethanegroups, epoxides, and ester groups.

Examples of peptide linkers as disclosed herein include the followingsequences: GSG, GGSG (SEQ ID NO: 2), GGGSG (SEQ ID NO: 3), GGGGSG (SEQID NO: 4), GGGGGSG (SEQ ID NO: 5), and GGGGGGSG (SEQ ID NO: 6) (see,e.g., experiments shown on FIGS. 2-5 herein). Other examples contemplateeven longer sequences that result from the sequential coupling ofadditional glycine monomeric subunits or other amino acid subunits tothe growing peptide linker.

In some embodiments, a linker as disclosed herein can maintain a netpositive charge at a pH of about 0, a pH of about 1, a pH of about 2, apH of about 3, a pH of about 4, a pH of about 5, a pH of about 6, a pHof about 7, a pH of about 8, a pH of about 9, a pH of about 10, a pH ofabout 11, a pH of about 12, a pH of about 13, or a pH of about 14.

In some embodiments, a linker as disclosed herein can maintain a netnegative charge at a pH of about 0, a pH of about 1, a pH of about 2, apH of about 3, a pH of about 4, a pH of about 5, a pH of about 6, a pHof about 7, a pH of about 8, a pH of about 9, a pH of about 10, a pH ofabout 11, a pH of about 12, a pH of about 13, or a pH of about 14.

In some embodiments, a linker as disclosed herein can maintain a netneutral charge at a pH of about 0, a pH of about 1, a pH of about 2, apH of about 3, a pH of about 4, a pH of about 5, a pH of about 6, a pHof about 7, a pH of about 8, a pH of about 9, a pH of about 10, a pH ofabout 11, a pH of about 12, a pH of about 13, or a pH of about 14.

In some embodiments, a linker as disclosed herein can maintain a netpositive charge at a pH above 0 and up to and including 1, a pH above 1and up to and including 2, a pH above 2 and up to and including 3, a pHabove 3 and up to and including 4, a pH above 4 and up to and including5, a pH above 5 and up to and including 6, a pH above 6 and up to andincluding 7, a pH above 7 and up to and including 8, a pH above 8 and upto and including 9, a pH above 9 and up to and including 10, a pH above10 and up to and including 11, a pH above 11 and up to and including 12,a pH above 12 and up to and including 13, or a pH above 13 and up to andincluding 14. In some embodiments, a linker can maintain a net negativecharge at a pH above 0 and up to and including 1, a pH above 1 and up toand including 2, a pH above 2 and up to and including 3, a pH above 3and up to and including 4, a pH above 4 and up to and including 5, a pHabove 5 and up to and including 6, a pH above 6 and up to and including7, a pH above 7 and up to and including 8, a pH above 8 and up to andincluding 9, a pH above 9 and up to and including 10, a pH above 10 andup to and including 11, a pH above 11 and up to and including 12, a pHabove 12 and up to and including 13, or a pH above 13 and up to andincluding 14.

In some embodiments, a linker as disclosed herein can maintain a netneutral charge at a pH above 0 and up to and including 1, a pH above 1and up to and including 2, a pH above 2 and up to and including 3, a pHabove 3 and up to and including 4, a pH above 4 and up to and including5, a pH above 5 and up to and including 6, a pH above 6 and up to andincluding 7, a pH above 7 and up to and including 8, a pH above 8 and upto and including 9, a pH above 9 and up to and including 10, a pH above10 and up to and including 11, a pH above 11 and up to and including 12,a pH above 12 and up to and including 13, or a pH above 13 and up to andincluding 14.

In some embodiments, a linker as disclosed herein can reduce thebackground signal on an array by at least 1%, at least 2%, at least 3%,at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 99%.

In some embodiments, a linker as disclosed herein can increase thesensitivity of the array by at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%.

In some embodiments, a linker as disclosed herein increases thesensitivity of the array by at least 1-fold, at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least15-fold.

In some embodiments, a linker as disclosed herein increases thespecificity of the array, as compared to an array with a decreasedlength or shorter linker, by at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%.

In some embodiments, a linker as disclosed herein increases thespecificity of the array, as compared to an array with a decreasedlength or shorter linker, by at least 1-fold, at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or at least15-fold.

In some embodiments, a linker as disclosed herein increases thereproducibility of the array, as compared to an array with a decreasedlength or shorter linker, by at least 1%, at least 2%, at least 3%, atleast 4%, at least 5%, at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%.

In some embodiments, a linker as disclosed herein increases thereproducibility of the array by at least 1-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or atleast 15-fold.

In some embodiments, the linker as disclosed herein increases the signalto background ratio of an array by at least 1%, at least 2%, at least3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%,at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%.

In some embodiments, the linker as disclosed herein increases the signalto background ratio of an array by at least 1-fold, at least 2-fold, atleast 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or atleast 15-fold.

In some embodiments, a linker as disclosed herein has a length of 2monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers, 7 monomers, 8monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18monomers, 19 monomers, 20 monomers, 21 monomers, 22 monomers, 23monomers, 24 monomers, 25 monomers, 26 monomers, 27 monomers, 28monomers, 29 monomers, 30 monomers, 31 monomers, 32 monomers, 33monomers, 34 monomers, 35 monomers, 36 monomers, 37 monomers, 38monomers, 39 monomers, 40 monomers, 41 monomers 42 monomers, 43monomers, 44 monomers, 45 monomers, 46 monomers, 47 monomers, 48monomers, 49 monomers, or 50 monomers.

In some embodiments, a linker as disclosed herein has a length between 2and 5 monomers, between 6 and 10 monomers, between 11 and 15 monomers,between 16 and 20 monomers, between 21 and 25 monomers, between 26 and30 monomers, between 31 and 35 monomers, between 36 and 40 monomers,between 41 and 45 monomers, between 46 and 50 monomers, between 51 and55 monomers, between 56 and 60 monomers, between 61 and 65 monomers,between 66 and 70 monomers, between 71 and 75 monomers, between 76 and80 monomers, between 81 and 85 monomers, between 86 and 90 monomers,between 91 and 95 monomers, or between 95 and 100 monomers.

In some embodiments, a linker as disclosed herein has a length of atleast 2 monomers, 3 monomers, 4 monomers, 5 monomers, 6 monomers, 7monomers, 8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers,13 monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18monomers, 19 monomers, 20 monomers, 21 monomers, 22 monomers, 23monomers, 24 monomers, 25 monomers, 26 monomers, 27 monomers, 28monomers, 29 monomers, 30 monomers, 31 monomers, 32 monomers, 33monomers, 34 monomers, 35 monomers, 36 monomers, 37 monomers, 38monomers, 39 monomers, 40 monomers, 41 monomers 42 monomers, 43monomers, 44 monomers, 45 monomers, 46 monomers, 47 monomers, 48monomers, 49 monomers, or 50 monomers.

In some embodiments, a particular linker sequence as disclosed herein isbetween 1 and 3 monomers in length, or between 4 and 6 monomers inlength, or between 7 and 9 monomers in length, or between 10 and 12monomers in length, or between 13 and 15 monomers in length, or between16 and 18 monomers in length, or between 19 and 21 monomers in length,or between 22 and 24 monomers in length, or between 25 and 27 monomersin length, or between 28 and 30 monomers in length, or between 31 and 33monomers in length, or between 34 and 36 monomers in length, or between37 and 39 monomers in length, or between 40 and 42 monomers in length,or between 43 and 45 monomers in length, or between 46 and 48 monomersin length, or between 49 and 51 monomers in length, or between 52 and 54monomers in length, or between 55 and 57 monomers in length, or between58 and 60 monomers in length, or between 61 and 63 monomers in length,or between 64 and 66 monomers in length, or between 67 and 69 monomersin length, or between 70 and 72 monomers in length, or between 73 and 75monomers in length, or between 76 and 78 monomers in length, or between79 and 81 monomers in length, or between 82 and 84 monomers in length,or between 85 and 87 monomers in length, or between 88 and 90 monomersin length, or between 91 and 93 monomers in length, or between 94 and 96monomers in length, or between 97 and 99 monomers in length, or between100 and 102 monomers in length.

In some embodiments, a particular functional sequence as disclosedherein has a length of 1 monomer, 2 monomers, 3 monomers, 4 monomers, 5monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10 monomers,11 monomers, 12 monomers, 13 monomers, 14 monomers, 15 monomers, 16monomers, 17 monomers, 18 monomers, 19 monomers, or 20 monomers.

In some embodiments, a particular functional sequence as disclosedherein has a length of at least 1 monomer, 2 monomers, 3 monomers, 4monomers, 5 monomers, 6 monomers, 7 monomers, 8 monomers, 9 monomers, 10monomers, 11 monomers, 12 monomers, 13 monomers, 14 monomers, 15monomers, 16 monomers, 17 monomers, 18 monomers, 19 monomers, or 20monomers.

In some embodiments, a particular functional sequence as disclosedherein is between 1 and 3 monomers in length, or between 4 and 6monomers in length, or between 7 and 9 monomers in length, or between 10and 12 monomers in length, or between 13 and 15 monomers in length, orbetween 16 and 18 monomers in length, or between 19 and 21 monomers inlength, or between 22 and 24 monomers in length, or between 25 and 27monomers in length, or between 28 and 30 monomers in length, or between31 and 33 monomers in length, or between 34 and 36 monomers in length,or between 37 and 39 monomers in length, or between 40 and 42 monomersin length, or between 43 and 45 monomers in length, or between 46 and 48monomers in length.

In some embodiments, a combined linker and sequence as disclosed hereinhas a length of at least 4 monomers, 5 monomers, 6 monomers, 7 monomers,8 monomers, 9 monomers, 10 monomers, 11 monomers, 12 monomers, 13monomers, 14 monomers, 15 monomers, 16 monomers, 17 monomers, 18monomers, 19 monomers, or 20 monomers.

In some embodiments, a combined linker and functional sequence asdisclosed herein is between 1 and 3 monomers in length, or between 4 and6 monomers in length, or between 7 and 9 monomers in length, or between10 and 12 monomers in length, or between 13 and 15 monomers in length,or between 16 and 18 monomers in length, or between 19 and 21 monomersin length, or between 22 and 24 monomers in length, or between 25 and 27monomers in length, or between 28 and 30 monomers in length, or between31 and 33 monomers in length, or between 34 and 36 monomers in length,or between 37 and 39 monomers in length, or between 40 and 42 monomersin length, or between 43 and 45 monomers in length, or between 46 and 48monomers in length.

In some embodiments, the linker sequences as disclosed herein present ona surface are of a uniform length. For example, in certain embodiments,photolithography masks may not be utilized, wherein linker synthesis isconducted upon the entire array surface to produce linkers of uniformlength. In other embodiments, the linker sequences present on a surfaceas disclosed herein are not of uniform length. For example, non-uniformlinker sequences on a surface may arise from the use of photolithographymasks during synthesis that result in differential addition of monomericsubunits to distinct features on the array surface. In certainembodiments, the linkers within a given feature can be of uniformlength, while having a distinct length from linkers in a differentfeature.

In some embodiments, the linker sequences as disclosed herein will be ofone uniform length for a subset of features and of a different uniformlength for at least one other subset of features on the given surface.

Surfaces

A surface as disclosed herein can refer to a material or group ofmaterials having rigid or semi-rigid properties. A surface can compriseone or more materials having porous, permeable, or semi-permeableproperties. A surface can also comprise one or more materials havingmalleable, ductile, brittle, plastic or elastic properties. A surfacecan also comprise one or more materials having polar, charged, acidic,or basic properties.

A surface as disclosed herein can be a flat surface, or a round orcurved surface (such as a bead). In certain embodiments of a flatsurface, the shape of that surface may be a circle, an oval, an ellipse,a crescent, a triangle, a curvlinear triangle, a quatrefoil, aparallelogram, a square, a rhombus, a trapezoid, a kite, a pentagon, ahexagon, a heptagon, an octagon, a nonagon, or a decagon. In certainembodiments of a round or curved surface, the surface may take on theshape of a sphere, an ellipsoid, or a more complex shape. In someembodiments, at least one surface of the solid support can besubstantially flat.

A surface as disclosed herein can be homogenous or heterogeneous. Asurface can comprise physical elements such as wells, raised regions,bumps, indentations, towers, pins, etched trenches, or combinationsthereof. In certain embodiments, the physical elements on the surfacecan be of roughly uniform dimensions. In certain embodiments, thephysical elements on the surface can be of non-uniform dimensions. Incertain embodiments, the physical elements occur on the surface atdiscrete locations in a non-repetitive pattern. In certain embodiments,the physical elements occur on the surface in a repetitive pattern. Insuch embodiments, repetitive physical elements that are adjacent to oneanother can be separated by a uniform distance. Alternatively, therepetitive physical elements that are adjacent to one another can beseparated by non-uniform distances.

In some embodiments, the solid support may be porous.

Non-limiting examples of substrate or surface materials as disclosedherein include, for example, silicon, biocompatible polymers such as,for example poly(methyl methacrylate) (PMMA) and polydimethylsiloxane(PDMS), glass, SiO, (such as, for example, a thermal oxide silicon wafersuch as that used by the semiconductor industry), quartz, siliconnitride, functionalized glass, gold, platinum, metalloporphyrins,tungsten, silica, diamond, titanium, polyester, polyamide, polyimide,polyether, polysulfone, fluoropolymer, and aluminum.

In certain embodiments, as disclosed herein an organosilane is used toprovide the surface attachment group. In certain embodiments, theorganosilane comprises aminosilane, epoxysilane, or fluorosilane.

Non-limiting examples of functionalized surfaces as disclosed hereininclude: amino-functionalized glass, carboxy functionalized glass,hydroxy functionalized glass, aldehyde functionalized glass,Streptavidin functionalized glass, Neutravidin functionalized glass,Avidin functionalized glass, Poly-L-Lysine functionalized glass,pyridine functionalized glass, and 3-amnopropyl-triethoxysilane (APTS)functionalized glass.

Additionally, a surface may optionally be coated with one or more layersto provide a surface for molecular attachment or functionalization,increased or decreased reactivity, binding detection, or otherspecialized application. In such embodiments, as disclosed herein thesurface may be optionally coated with at least 1 layer, at least 2layers, at least 3 layers, at least 4 layers, at least 5 layers, atleast 6 layers, at least 7 layers, at least 8 layers, at least 9 layers,at least 10 layers, at least 11 layers, at least 12 layers, at least 13layers, at least 14 layers, at least 15 layers, at least 16 layers, atleast 17 layers, at least 18 layers, at least 19 layers, or at least 20layers. In certain embodiments, the layers coated on the surface canprovide cumulative properties such as increased molecular attachment orfunctionalization, increased or decreased reactivity, binding detection,or other specialized application. In certain embodiments, the layerscoated on the surface can provide discrete properties such as one layerproviding molecular attachment for one category of monomers or polymerswhile another layer provides molecular attachment for a differentcategory of monomers or polymers. For example, one layer may providemolecular attachment for peptides, while a second layer providesmolecular attachment for nucleic acids to form a combination array.

Substrate or surface materials and or layer(s) may be porous ornon-porous. For example, a substrate may be comprised of porous silicon.Furthermore, the substrate or surface materials may be permeable orsemipermeable. Additionally, the substrate as disclosed herein may be asilicon wafer or chip such as those used in the semiconductor devicefabrication industry. In the case of a wafer or chip, a plurality ofarrays may be synthesized on the wafer. In some embodiments, thesubstrate is chosen from glass, silicon and silicon having a siliconoxide layer.

Methods of Synthesis

In some embodiments as disclosed herein, a method for in situ synthesisof the array is photolithography-based. In some embodiments, thephotolithography-based synthesis comprises a photomask patterned step.

In some embodiments as disclosed herein, a method for thephotolithography-based synthesis of the arrays uses a minimum number ofphotomasks to construct the array. In some embodiments, the arraycomprises features of about 0.5 micron to about 200 microns in diameterand a center-to-center distance of about 1 micron to about 300 micronson center.

In some embodiments as disclosed herein, a feature is about 0.5 micronto about 10 microns in diameter; about 11 microns to about 20 microns indiameter; about 21 microns to about 30 microns in diameter; about 31microns to about 40 microns in diameter; about 41 microns to about 50microns in diameter; about 51 microns to about 60 microns in diameter;about 61 microns to about 70 microns in diameter; about 71 microns toabout 80 microns in diameter; about 81 microns to about 90 microns indiameter; about 91 microns to about 100 microns in diameter; about 101microns to about 110 microns in diameter; about 111 microns to about 120microns in diameter; about 121 microns to about 130 microns in diameter;about 131 microns to about 140 microns in diameter; about 141 microns toabout 150 microns in diameter; about 151 microns to about 160 microns indiameter; about 161 microns to about 170 microns in diameter; about 171microns to about 180 microns in diameter; about 181 microns to about 190microns in diameter; about 191 microns to about 200 microns in diameter.

In some embodiments as disclosed herein, the center-to-center distancebetween adjacent features is about 1 micron to about 10 microns; about11 microns to about 20 microns; about 21 microns to about 30 microns;about 31 microns to about 40 microns; about 41 microns to about 50microns; about 51 microns to about 60 microns; about 61 microns to about70 microns; about 71 microns to about 80 microns; about 81 microns toabout 90 microns; about 91 microns to about 100 microns; about 101microns to about 110 microns; about 111 microns to about 120 microns;about 121 microns to about 130 microns; about 131 microns to about 140microns; about 141 microns to about 150 microns; about 151 microns toabout 160 microns; about 161 microns to about 170 microns; about 171microns to about 180 microns; about 181 microns to about 190 microns;about 191 microns to about 200 microns; about 201 microns to about 210microns; about 211 microns to about 220 microns; about 221 microns toabout 230 microns; about 231 microns to about 240 microns; about 241microns to about 250 microns; about 251 microns to about 260 microns;about 261 microns to about 270 microns; about 271 microns about 280microns; about 281 microns to about 290 microns; about 291 microns toabout 300 microns.

In some embodiments as disclosed herein, the array comprises at least10,000 features, at least 20,000 features, at least 30,000 features, atleast 40,000 features, at least 50,000 features, at least 60,000features, at least 70,000 features, at least 80,000 features, at least90,000 features, at least 100,000 features, at least 110,000 features,at least 120,000 features, at least 130,000 features, at least 140,000features, at least 150,000 features, at least 160,000 features, at least170,000 features, at least 180,000 features, at least 190,000 features,at least 200,000 features, at least 210,000 features, at least 220,000features, at least 230,000 features, at least 240,000 features, at least250,000 features, at least 260,000 features, at least 270,000 features,at least 280,000 features, at least 290,000 features, at least 300,000features, at least 310,000 features, at least 320,000 features, at least330,000 features, at least 340,000 features, at least 350,000 features,at least 360,000 features, at least 370,000 features, at least 380,000features, at least 390,000 features, at least 400,000 features, at least410,000 features, at least 420,000 features, at least 430,000 features,at least 440,000 features, at least 450,000 features, at least 460,000features, at least 470,000 features, at least 480,000 features, at least490,000 features, at least 500,000 features, at least 600,000 features,at least 700,000 features, at least 800,000 features, at least 900,000features, or at least 1,000,000 features.

In some embodiments as disclosed herein, the surface of the array isconditioned or pre-conditioned prior to attachment of the sequences ontothe surface of the array. Conditioning of the array surface includesexposing the features on the surface to the coupling and photoresistsolutions in the absence of monomers to form sequences.

In some embodiments as disclosed herein, at least one pre-conditioningor conditioning step is performed on the surface prior to the mainsynthesis of the functional sequence.

In some embodiments as disclosed herein, the conditioning step isperformed at least 1 time, 2 times, 3 times, 4 times, 5 times, 6 times,7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14times, 15 times, 16 times, 17 times, 18 times, 19 times, 20 times, 21times, 22 times, 23 times, 24 times, 25 times, 26 times, 27 times, 28times, 29 times, or 30 times.

In some embodiments as disclosed herein, the conditioning step isperformed no more than 1 time, 2 times, 3 times, 4 times, 5 times, 6times, 7 times, 8 times, 9 times, 10 times, 11 times, 12 times, 13times, 14 times, 15 times, 16 times, 17 times, 18 times, 19 times, 20times, 21 times, 22 times, 23 times, 24 times, 25 times, 26 times, 27times, 28 times, 29 times, or 30 times.

In some embodiments as disclosed herein, the conditioning step isperformed 1 time, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times,8 times, 9 times, 10 times, 11 times, 12 times, 13 times, 14 times, 15times, 16 times, 17 times, 18 times, 19 times, 20 times, 21 times, 22times, 23 times, 24 times, 25 times, 26 times, 27 times, 28 times, 29times, or 30 times.

In some embodiments, conditioning the surface of the array as disclosedherein can increase the sensitivity of the array by at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 100%.

In some embodiments, conditioning the surface of the array as disclosedherein can increase the sensitivity of the array by at least 1-fold, atleast 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, atleast 10-fold, or at least 15-fold.

In some embodiments, conditioning the surface of the array as disclosedherein can increase the specificity of the array, as compared to anarray with a decreased length or shorter linker, by at least 1%, atleast 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or at least 100%.

In some embodiments, conditioning the surface of the array as disclosedherein can increase the specificity of the array, as compared to anarray in the absence of conditioning, at least 1-fold, at least 2-fold,at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, atleast 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, or atleast 15-fold.

In some embodiments, conditioning the surface of the array as disclosedherein can increase the reproducibility of the array, as compared to anarray in the absence of conditioning, by at least 1%, at least 2%, atleast 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least20%, at least 25%, at least 30%, at least 35%, at least 40%, at least45%, at least 50%, at least 55%, at least 60%, at least 65%, at least70%, at least 75%, at least 80%, at least 85%, at least 90%, at least95%, or at least 100%.

In some embodiments, conditioning the surface of the array as disclosedherein can increase the reproducibility of the array by at least 1-fold,at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, atleast 10-fold, or at least 15-fold.

In yet other embodiments, conditioning the surface of the array asdisclosed herein can enhance binding of a target to the sequence on thearray by at least 1%, at least 2%, at least 3%, at least 4%, at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 35%, at least 40%, at least 45%, at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, or at least 100%.

In still other embodiments, conditioning the surface of the array asdisclosed herein can enhance binding of a target to the sequence on thearray by at least 1-fold, at least 2-fold, at least 3-fold, at least4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least8-fold, at least 9-fold, at least 10-fold, or at least 15-fold.

EXAMPLES Example 1: Multiple Successive Additions of a Specific Epitope

Steps (1) through (4) describe the generation of an array with multiplesuccessive additions of a specific epitope:

Step (1). An 8 inch silicon wafer with a 2500 angstrom thermal oxidelayer was cleaned extensively with a solution of sulfuric acid (83.5% involume) and 30% hydrogen peroxide (16.5% in volume). This surface wasextensively flushed with highly purified water. The wafer was thenplaced in a 0.5% solution by volume of aminopropyltriethoxysilane in 95%ethanol and 4.5% water and allowed to sit without agitation for 90minutes. The wafer was next washed in ethanol and isopropanol,spin-dried and placed in an oven at 110° C. for 90 minutes. The waferwas then stored under nitrogen for two days. Then the wafer was placedon a spin coater and a 10 mL coupling solution of Boc-Gly (0.1M),6-Cl-HOBT (0.1M) and diisopropylcarbodiimide (DIC, 0.1M) inN-methylpyrrolidinone (NMP) was added while spinning at 500 RPM. Thewafer was then removed from the spin coater and a second clean wafer wasplaced on top of it in such a way that the coupling solution spun ontoit was trapped between the two wafers. This arrangement was placed on ahot plate at 85° C. for 120 seconds. The cover plate was removed, thewafer was placed back on the spin coater and 10 mL of NMP was added asthe wafer was spun at 2000 RPM to wash off the coupling solution. Thiscoupling was repeated one more time before it was washed on a spincoater with solvents in order of 10 mL N-methylpyrrolidinone (NMP), 10mL methylisobutylketone (MIBK), and 10 mL isopropanol (IPA) at 2000RPM.

Step (2). The wafer was then placed on a spin coater and 10 mL of aphotoresist solution (25 mg/ml poly methyl methacrylate (PMMA), 30 mg/mlBis(4-t-butylphenyl)iodonium triflate (PAG), and 15 mg/mLisopropyl-9H-thioxanthen-9-one (ITX) in propylene glycol methyl etheracetate (PGMEA)) were added while spinning at 2000 RPM. The wafer wasthen kept at 75° C. for 1.5 minutes. The wafer was placed on an alignerand aligned to a precise position relative to a mask (withinapproximately 1-2 microns in the two horizontal dimensions and 10microns in the vertical dimension). The mask was designed to exposecertain features on the wafer to UV light centered at 365 nm. The waferwas exposed through the mask for 7 seconds using a total exposure powerof 19 milliwatts/cm². The wafer was again placed on the spin coater, and10 mL of methylisobutylketone (MIBK) was added while spinning at 2000RPM to remove the photoresist. This was followed by 10 mL of isopropanolat the same speed. 10 mL of a coupling solution consisting of Boc-Ser(0.1M), DIC (0.1M), and 6-Cl-HOBT (0.1M) in NMP was then added whilespinning at 500 RPM. The wafer was then removed from the spin coater anda second clean wafer was placed on top of it in such a way that thecoupling solution spun onto it was trapped between the two wafers. Thisarrangement was placed on a hot plate at 85° C. for 120 seconds. Thecover plate was removed, the wafer placed back on the spin coater and 10mL of NMP was added as the wafer was spun at 2000 RPM to wash off thecoupling solution. 10 mL of MIBK was then added as the wafer was spun at2000 RPM, followed by 10 mL IPA at the same speed.

Step (3). The process described above in (2) was repeated 21 times, eachtime using an appropriate mask and amino acid. The amino acids wereadded in the following order: first=V; second=V; third=S; fourth=H;fifth=R; sixth=V; seventh=V; eight=S; ninth=H; tenth=R; eleventh=V;twelfth=V; thirteenth=S; fourteenth=H; fifteenth=R; sixteenth=V;seventeenth=V; eighteenth=S; nineteenth=H; twentieth=R; and each aminoacid was added in same concentration of 0.1M as shown in (2). The oneletter designation of each amino acid is used in the foregoing and thatdesignation is well known to those skilled in the art.

Step (4). The wafer described above was diced in such away that thearrays of ˜330,000 peptides could be individually exposed to specificsamples. In this way arrays were incubated with 5 pM of a monoclonalantibody P53Ab1. There were some peptides with the sequence RHSVVGSG(SEQ ID NO: 7) on the array. In some cases the RHSVV (SEQ ID NO: 1) partof the peptide was synthesized early in the synthesis (designatedRHSVVaGSG (SEQ ID NO: 7) in FIG. 1 ), some successively later during thesynthesis (designated RHSVVbGSG (SEQ ID NO: 7), RHSVVcGSG (SEQ ID NO:7), and RHSVVdGSG (SEQ ID NO: 7)).

Example 2: Multiple Successive Additions of a Specific Epitope

The binding of the P53Ab1 monoclonal antibody to an array comprisingeach of the peptides described in EXAMPLE 1 was determined and is shownin FIG. 1 . FIG. 1 illustrates the results of the surface stability testperformed upon peptides synthesized successively. Each of the sequenceson the x-axis was generated at successively later times during thesynthesis. The y-axis shows the intensity of binding of the P53Ab1monoclonal antibody to RHSVV sequences (SEQ ID NO: 1) that weresynthesized early in the synthesis (labeled RHSVVaGSG (SEQ ID NO: 7) inFIG. 1 ), and to sequences that were synthesized later during thesynthesis (labeled RHSVVbGSG (SEQ ID NO: 7), RHSVVcGSG (SEQ ID NO: 7),and RHSVVdGSG (SEQ ID NO: 7) in FIG. 1 ). The data suggests that thelater the functional sequence is synthesized in the process, the greaterthe intensity of binding to a target. This suggests that even though afeature is not having an amino acid added to it, the process of exposingthe features on the surface to the coupling and the photoresistsolutions increases the amount of peptide made that binds P53Ab1. Inother words, the early synthetic cycles in the absence of an amino acidmonomer precondition the feature for enhanced in situ synthesis. FIG. 1illustrates an approximately 3-fold enhancement of binding whencomparing the RHSVVaGSG (SEQ ID NO: 7) and RHSVVdGSG (SEQ ID NO: 7)peptides. This is surprising particularly because no additional monomerswere added to the array between the pre-conditioning steps.

Example 3: Varying the Linker Length and Composition Before SuccessiveAddition of a Specific Epitope

Step (1). An 8 inch silicon wafer with a 2,500 angstrom thermal oxidelayer was cleaned extensively with a solution of sulfuric acid (83.5% involume) and 30% hydrogen peroxide (16.5% in volume). This surface wasextensively flushed with highly purified water. The wafer was thenplaced in a 0.5% solution by volume of aminopropyltriethoxysilane in 95%ethanol and 4.5% water and allowed to sit without agitation for 90minutes. The wafer was next washed in ethanol and isopropanol and thenplaced in an oven at 110° C. for 90 minutes. The wafer was then storedunder nitrogen for two days. Then the wafer was placed on a spin coater,and a 10 mL coupling solution of Boc-Gly (0.1M), 6-Cl-HOBT (0.1M), anddiisopropylcarbodiimide (DIC) in N-methylpyrrolidinone (NMP) was addedwhile spinning at 500 RPM. The wafer was then removed from the spincoater and a second clean wafer was placed on top of it in such a waythat the coupling solution spun onto it was trapped between the twowafers. This arrangement was placed on a hot plate at 85° C. for 120seconds. The cover plate was removed, the wafer placed back on the spincoater and 10 ml of NMP was added as the wafer was spun at 2000 RPM towash off the coupling solution. The coupling was repeated one more timebefore it was washed on a spin coater in order of 10 mL NMP, 10 mL MIBK,and 10 mL IPA, each at 2000 RPM.

Step (2) The wafer was then placed on a spin coater and 10 mL of aphotoresist solution (25 mg/ml poly methyl methacrylate (PMMA), 30 mg/mlBis(4-t-butylphenyl)iodonium triflate (PAG) and 15 mg/mLisopropyl-9H-thioxanthen-9-one (ITX) in propylene glycol methyl etheracetate (PGMEA)) were added while spinning at 2,000 RPM. The wafer wasthen placed on a hot place at 75° C. for 1.5 minutes. The wafer wasplaced on an aligner and aligned to a precise position relative to amask (within approximately 1-2 microns in the two horizontal dimensionsand 10 microns in the vertical dimensions). The mask was designed toexpose certain features on the wafer to UV light centered at 365 nm. Thewafer was exposed through the mask for 7 seconds using a total exposurepower of 19 milliwatts/cm². The wafer was again placed on the spincoater and 10 mL of methylisobutylketone was added while spinning at2,000 RPM to remove the photoresist. This was followed by 10 mL ofisopropanol at the same speed. 10 mL of a coupling solution consistingof Boc-Ser (0.1M), DIC (0.1M), and 6-Cl-HOBT (0.1M) in NMP was thenadded while spinning at 500 RPM. The wafer was then removed from thespin coater and a second clean wafer was placed on top of it in such away that the coupling solution spun onto it was trapped between the twowafers. This arrangement was placed on a hot plate at 85° C. for 120seconds. The cover plate was removed, the wafer placed back on the spincoater and 10 mL of NMP was added as the wafer was spun at 2,000 RPM towash off the coupling solution. 10 mL of MIBK was then added as thewafer was spun at 2,000 RPM, followed by 10 mL IPA at the same speed toclean and dry the wafer.

Step (3) The process described above in (2) was repeated a number oftimes, each time using an appropriate mask and amino acid. The aminoacids were added in the order: first=G; second=G; third=S; forth=G;fifth=G; sixth=S; seventh=G; eight=G; ninth=S; tenth=G; eleventh=G;twelfth=S; thirteenth=G; fourteenth=G; fifteenth=S; sixteenth=G;seventeenth=G; eighteenth=S; nineteenth=G; twentieth=G; twenty-first=S;twenty-second=G; twenty-third=V; twenty-fourth=V; twenty-fifth=S;twenty-six=H; twenty-seventh=R; twenty-eight=V; twenty-ninth=V;thirtieth=S; thirty-first=H; thirty-second=R; thirty-third=V;thirty-fourth=V; thirty-fifth=S; thirty-six=H; thirty-seventh=R;thirty-eighth=V; thirty-ninth=V; fortieth=S; fortieth-first=H; andfortieth-second=R. The masks were designed such that the number of G'sand S's varied from one peptide to another, but all peptides ended withRHSVV (SEQ ID NO: 1) (the epitope for P53Ab1). All amino acids wereadded in the same concentration (0.1M) as shown in (2). The one letterdesignation of each amino acid is used in the foregoing and thatdesignation is well known to those skilled in the art.

Step (4) The wafer described above was diced in such away that thearrays of ˜330,000 peptides could be individually exposed to specificsamples as described previously (Legutki et al., 2014, NatureCommunications, 5, Article number: 4785 doi:10.1038/ncomms5785). In thisway arrays were incubated with 5.0 pM of a monoclonal antibody P53Ab1.

FIG. 2 illustrates the results of the surface stability test performedupon peptides synthesized successively on a seven amino acid linker.FIG. 2 illustrates a linker that was synthesized the surface and thenRHSVV (SEQ ID NO: 1) was synthesized four times at different places. Theproduction of the linker involved 24 synthetic cycles. The y-axis showsthe intensity of binding of the P53Ab1 monoclonal antibody to RHSVVsequences (SEQ ID NO: 1) that were synthesized early in the synthesis(labeled RHSVVaGSG (SEQ ID NO: 7) in FIG. 2 ), and to sequences thatwere synthesized one cycle later (labeled RHSVVbGSG (SEQ ID NO: 7) inFIG. 2 ), two cycles later (labeled RHSVVcGSG (SEQ ID NO: 7) in FIG. 2), or three cycles later (labeled RHSVVdGSG (SEQ ID NO: 7) in FIG. 2 ).As illustrated in FIG. 2 , the longer linker (GGGGGSG (SEQ ID NO: 5))gives almost 10 times more P53Ab1 signal overall than the GSG linker(FIG. 1 ), which is a surprising and unexpected increase in bindingstrength considering the mere length difference of four amino acids. Inaddition, the change in binding as a function of when the epitope wasmade is minimal for peptides with the GGGGSG linker (SEQ ID NO: 4).

FIG. 3 illustrates the results of the surface stability test performedupon peptides synthesized successively on a three amino acid linker. They-axis shows the intensity of binding of the P53Ab1 monoclonal antibodyto RHSVV sequences (SEQ ID NO: 1) that were synthesized early in thesynthesis (labeled RHSVVaGSG (SEQ ID NO: 7) in FIG. 3 ), and tosequences that were synthesized one cycle later (labeled RHSVVbGSG (SEQID NO: 7) in FIG. 3 ), two cycles later (labeled RHSVVcGSG (SEQ ID NO:7) in FIG. 3 ), or three cycles later (labeled RHSVVdGSG (SEQ ID NO: 7)in FIG. 3 ). FIG. 4 illustrates that longer linkers result in highbinding of the P53Ab1 to the surface.

Example 4: Application of the Immunosignature Approach to Detection andIdentification of Infectious Disease

Step (1). An 8 inch silicon wafer with a 2,500 angstrom thermal oxidelayer was cleaned extensively with a solution of sulfuric acid (83.5% byvolume) and 30% hydrogen peroxide (16.5% by volume). This surface wasextensively flushed with highly purified water. The wafer was thenplaced in a 0.5% solution by volume of aminopropyltriethoxysilane in 95%ethanol and 4.5% water and allowed to sit without agitation for 90minutes. The wafer was next washed in ethanol and isopropanol and placedin an oven at 110° C. for 90 minutes. Then the wafer was placed on aspin coater, and a 10 mL coupling solution of Boc-Gly (0.1M), 6-Cl-HOBT(0.1M), and diisopropylcarbodiimide (DIC, 0.1M) in N-methylpyrrolidinone(NMP) was added while spinning at 500 RPM. The wafer was then removedfrom the spin coater and a second clean wafer was placed on top of it insuch a way that the coupling solution spun onto it was trapped betweenthe two wafers. This arrangement was placed on a hot plate at 85° C. for120 seconds. The cover plate was removed, the wafer placed back on thespin coater and 10 mL of NMP was added as the wafer was spun at 2,000RPM to wash off the coupling solution. 10 mL of MIBK was then added asthe wafer was spun at 2,000 RPM, followed by 10 mL IPA at the same speedto clean and dry the wafer. The coupling was repeated one more time.

Step (2). The wafer was placed on a spin coater and 10 mL of aphotoresist solution (25 mg/ml poly methyl methacrylate (PMMA), 30 mg/mlBis(4-t-butylphenyl)iodonium triflate (PAG) and 15 mg/mLisopropyl-9H-thioxanthen-9-one (ITX) in propylene glycol methyl etheracetate (PGMEA)) were added while spinning at 2,000 RPM. The wafer wasthen placed on a hot place at 75° C. for 1.5 minutes. The wafer wasplaced on an aligner and aligned to a precise position relative to amask (within approximately 1-2 microns in the two horizontal dimensionsand 10 microns in the vertical dimension). The mask was designed toexpose certain features on the wafer is UV light centered at 365 nm. Thewafer was exposed through the mask for 7 seconds using a total exposurepower of 19 milliwatts/cm². The wafer was again placed on the spincoater and 10 mL of methylisobutylketone was added while spinning at2,000 RPM to remove the photoresist. This was followed by 10 mL ofisopropanol at the same speed. 10 mL of a coupling solution consistingof Boc-Ser (0.1M), DIC (0.1M), and 6-Cl-HOBT (0.1M) in NMP was thenadded while spinning at 500 RPM. The wafer was then removed from thespin coater and a second clean wafer was placed on top of it in such away that the coupling solution spun onto it was trapped between the twowafers. This arrangement was placed on a hot plate at 85° C. for 120seconds. The cover plate was removed, the wafer placed back on the spincoater and 10 mL of NMP was added as the wafer was spun at 2,000 RPM towash off the coupling solution. 10 mL of MIBK was then added as thewafer was spun at 2,000 RPM, followed by 10 mL IPA at the same speed toclean and dry the wafer.

Step (3). The process described above in (2) was then repeated 18 timesexcept that the mask alignment and light exposure were removed from theprocess. These are the conditioning steps of the overall procedure butwith only one amino acid, Boc-Gly, used in the concentration of 0.1M asshown in (2).

Step (4). The wafer was then placed on a spin coater and 10 mL of aphotoresist solution (25 mg/ml poly methyl methacrylate (PMMA), 30 mg/mlBis(4-t-butylphenyl)iodonium triflate (PAG) and 15 mg/mLisopropyl-9Hthioxanthen-9-one (ITX) in propylene glycol methyl etheracetate (PGMEA)) were added while spinning at 2,000 RPM. The wafer wasthen placed on a hot place at 75° C. for 1.5 minutes. The wafer wasplaced on an aligner and aligned to a precise position relative to amask (within approximately 1-2 microns in the two horizontal dimensionsand 10 microns in the vertical dimension). The mask was designed toexpose certain features on the wafer to UV light centered at 365 nm. Thewafer was exposed through the mask for 7 seconds using a total exposurepower of 19 milliwatts/cm². The wafer was again placed on the spincoater and 10 mL of MIBK was added while spinning at 2,000 RPM to removethe photoresist. This was followed by 10 mL of IPA at the same speed. 10mL of a coupling solution consisting of Boc-Gly (0.1M), DIC (0.1M), and6-Cl-HOBT (0.1M) in NMP was then added while spinning at 500 RPM. Thewafer was then removed from the spin coater and a second clean wafer wasplaced on top of it in such a way that the coupling solution spun ontoit was trapped between the two wafers. This arrangement was placed on ahot plate at 85° C. for 120 seconds. The cover plate was removed, thewafer placed back on the spin coater and 10 mL of NMP was added as thewafer was spun at 2,000 RPM to wash off the coupling solution. 10 mL ofMIBK was then added as the wafer was spun at 2,000 RPM, followed by 10mL IPA at the same speed.

Step (5). The process described above in (4) was repeated 69 times, eachtime using a different mask and a different amino acid. The amino acidswere added in the order: first=G; second=G; third=G; fourth=R; fifth=L;sixth=Y; seventh=N; eight=H; ninth=S; tenth=D; eleventh=L; twelfth=W;thirteenth=K; fourteenth=E; fifteenth=H; sixteenth=K; seventeenth=W;eighteenth=D; nineteenth=V; twenty-first=Y; twenty-second=R;twenty-third=E; twenty-forth=F; twenty-fifth=P; twenty-sixth=V;twenty-seventh=F; twenty-eight=K; twenty-ninth; thirtieth=E;thirty-first=Q; thirty-second=N; thirty-third=Y; thirty-fourth=R;thirty-fifth=G; thirty-sixth=E; thirty-seventh=W; thirty-eight=D;thirty-ninth=G; fortieth=K; forty-first=Q; forty-second=L; forty-third;forty-fourth=Y; forty-fifth=V; forty-seventh=G; forty-eight=W;forty-ninth=E; fiftieth=G; fifty-first=S; fifty-second=P; fifty-third=R;fifty-fourth=V; fifty-fifth=P; fifty-sixth=H; fifty-seventh=R;fifty-eight=K; fifty-ninth=N; sixtieth=L; sixty-first=S; sixty-second=L;sixty-third=R; sixty-forth=A; sixty-fifth=D; sixty-eight=A;sixty-ninth=Y; seventieth=H; seventy-first=G; seventy-second=E;seventy-third=Q; seventy-forth=K; seventy-fifth=N). All amino acids wereadded in the same concentration of 0.1M. The one letter designation ofeach amino acid is used in the forgoing and that designation is wellknown to those skilled in the art.

Step (6). The wafer described above was diced such that arrays of˜330,000 peptides could be individually exposed to specific samples asdescribed previously. 24 blood samples from patients infected withMalaria, Dengue fever, West Nile Virus, or patients that were notinfected were analyzed for binding patterns and the immunosignature wasdetermined. A set of 50 peptides were chosen that provided the mostunique antibody binding data for each of the diseases. See Legutki etal., Nat. Commun. 5:4785 (2014). FIG. 5 is a visual representation of animmunosignature as a heat map. Each column in the heat map represents asample from an individual with a particular disease. Each row representsa peptide from the array. The color indicates the level of binding, withred being high binding and blue low binding. Note that for anyparticular disease, there is high binding only in a subset of peptides.That subset is distinct for different diseases.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A method for making an array comprising: a.performing a conditioning step on a surface of the array in an absenceof monomers, which conditioning step comprises exposing one or morefeatures on the surface to one or more coupling solutions and to one ormore photoresist solutions; b. repeating step (a) at least once; c.performing a synthesis step upon the surface to add at least onemonomer; and d. repeating step (c) at least once to form a sequence,wherein, as compared to an array made in an absence of performing steps(a) and (b), steps (a) and (b) performed prior to synthesizing thesequence enhance attachment of the sequence to the surface of the array,increase a sensitivity of the array by at least 1%, increase aspecificity of the array by at least 1%, increase a reproducibility ofthe array by at least 1%, and enhance binding of a target to thesequence on the array by at least 1%.